Thermo Fisher Scientific Rat IL-4 Matched Pair User guide

Rat IL-4 Matched Pair

Module Set for the development of an ELISA for quantitative detection of rat IL-4

Catalog Number BMS628MST

Pub. No. MAN0016901 Rev. A.0 (30)

WARNING! Read the Safety Data Sheets (SDSs) and follow the

handling instructions. Wear appropriate protective eyewear,

clothing, and gloves. Safety Data Sheets (SDSs) are available

from thermosher.com/support.

Read before opening

• Some vials contain small quantities of material, therefore

centrifuge before use.

• This set of reagents is intended for use by persons experienced in

the use of immunoassays. It is not suitable for use by

inexperienced personnel.

• A sample protocol is included, but the protocol provided is a

guideline. The type of substrate as well as all other reagents not

included in the Module Set may inuence assay performance.

Reagents provided

1 vial (1.1 mL) monoclonal Coating Antibody to rat IL-4 (100 µg/mL)

1 vial (55 µL) Biotin-Conjugate anti-rat IL-4 monoclonal antibody

1 vial (11 µL) Streptavidin-HRP

1 vial rat IL-4 Standard protein lyophilized, 2 ng/mL upon

reconstitution

2 vials (50 mL) Sample Diluent

Storage instructions

Store the kit components at 2°C to 8°C. Immediately after use

remaining reagents should be returned to cold storage (2°C to 8°C).

Aliquot reagents for repeated use at later dates. Reagents are labeled

with expiration date. For specic storage instructions see also

“Preparation of immunological reagents“ on page 2.

Samples should be aliquoted and must be stored frozen at -20°C to

avoid loss of bioactive rat IL-4.

Reagents and materials not provided

• Microwell plate

•Buers and solutions (see “Preparation of buers and

solutions“ on page 1 for preparation guidelines)

Precautions for use

All reagents should be considered as potentially hazardous. We

therefore recommend that this product is handled only by those

persons who have been trained in laboratory techniques and that it is

used in accordance with the principles of good laboratory practice.

Wear suitable protective clothing such as laboratory overalls, safety

glasses and gloves. Care should be taken to avoid contact with skin or

eyes. In the case of contact with skin or eyes wash immediately with

water. See material safety data sheet(s) for specic advice.

Preparation of buffers and solutions

Note: The quality of BSA is a critical parameter for the test

performance.

Phosphate buffered saline (PBS)

Reagents Quantity

NaCl 8.00 g

KCl 0.20 g

Na2HPO4 x 12 H2O 2.85 g

KH2PO40.20 g

H2O dest adjust to 1 liter

Wash buffer

Add 0.5 mL Tween™ 20 to 1 liter of PBS and mix well.

Assay buffer

Reagents Quantity

Bovine Serum Albumin (BSA) 5 g

Tween™ 20 0.5 mL

PBS adjust to 1 liter

Fixing buffer

Reagents Quantity

Sucrose 75 g

PBS adjust to 500 mL

Substrate solution

1:2 mixture of H2O2 and Tetramethylbenzidine

Stop solution

1M Phosporic Acid (H3PO4)

Preparation of the microwell plate

Coating

1. Coating antibody nal concentration is 1 µg/mL; 100 µL of the

coating solution is added to each well. Dilute the coating

antibody as following for one microtiter plate:

Reagents Volume

PBS 10.89 mL

Coating antibody (100 µg/mL) 0.11 mL

Coating solution (1 µg/mL) 11.00 mL

2. Immediately after coating, seal the plate with an adhesive lm

and store at 2°C to 8°C over night, allowing the binding process

to take place. Aspirate the contents of the wells and wash once

with 400 µL of Wash Buer according the washing procedure

described in the test protocol below (see “Test protocol“ on

page 2).

USER GUIDE

For Research Use Only. Not for use in diagnostic procedures.

Blocking and ﬁxing

Blocking

Add 250 µL of Assay Buer to each well and incubate at room

temperature for 2 hours. Alternatively the plate may be blocked over

night at 2°C to 8°C. Blocked plates can be stored at 2°C to 8°C for up to

one week.

Fixing

To store the coated plates for longer than one week aspirate Assay

Buer and add 150 µL Fixing Buer to each well. Incubate for 1 hour

at room temperature. Aspirate Fixing Buer and dry plates for 2 hours

at 37°C. When sealed with desiccant, the plates can be stored at 2°C to

8°C for 2 months.

Preparation of immunological reagents

Note: Centrifuge vials before opening to collect contents.

Preparation of standard

1. Reconstitute rat IL-4 standard protein with deionized or distilled

water. Reconstitution volume is indicated on the vial label (nal

concentration of reconstituted standard = 2 ng/mL). Allow the

standard to reconstitute for 10-30 minutes. Mix well prior to

making dilutions.

2. The concentrated rat IL-4 standard must be diluted 1:10 with

Assay Buer just prior to use in a clean plastic test tube according

to the following dilution scheme:

Reagents Volume

conc. Standard Protein (2 ng/mL) 23 µL

Assay Buffer 207 µL

Standard Protein (200 pg/mL) 230 µL

3. Shake gently to mix. After usage remaining diluted standard

cannot be stored and has to be discarded.

4. Aliquot the reconstituted concentrated standard and store at

-20°C.

Preparation of Biotin-Conjugate

Dilute concentrated Biotin-Conjugate 1:1000 with Assay Buer before

use. Use within 30 minutes after preparation. For one microwell plate

dilute the stock reagents as follows:

Reagents Volume

conc. Biotin-Conjugate 5.5 µL

Assay Buffer 5494.5 µL

Biotin-Conjugate 5.5 mL

Preparation of Streptavidin-HRP

Dilute concentrated Streptavidin-HRP 1:10,000 with Assay Buer

before use. Use within 30 minutes after preparation. For one microwell

plate dilute the stock reagents as follows:

Reagents Volume

conc. Streptavidin-HRP 1.1 µL

Assay Buffer 10,998.9 µL

Streptavidin-HRP 11.0 mL

Test protocol

1. Wash blocked or blocked and xed plates twice with

approximately 400 µL Wash Buer per well, with thorough

aspiration of microwell contents between washes. Allow the Wash

Buer to sit in the wells for about 10 – 15 seconds before

aspiration. Take care not to scratch the surface of the microwells.

After the last wash step, empty wells and tap microwell strips on

absorbent pad or paper towel to remove excess Wash Buer. Use

the microwell plate immediately after washing. Alternatively

microwell plate can be placed upside down on a wet absorbent

paper for no longer than 15 minutes. Do not allow wells to dry.

2. Add 100 µL Sample Diluent in duplicate all standard wells.

Pipee 100 µL of diluted standard (see “Preparation of

standard“ on page 2), (concentration = 200 pg/mL) in duplicate

into well A1 and A2. Mix the contents of wells A1 and A2 by

repeated aspiration and ejection (concentration of standard 1, S1 =

100 pg/mL), and transfer 100 µL to wells B1 and B2, respectively

(see Figure 1). Take care not to scratch the surface of the

microwells. Continue this procedure 5 times, creating two serially

diluted columns of rat IL-4 standard dilutions ranging from 100.0

to 1.60 pg/mL. Discard 100 µL from the last microwells (G1, G2).

Final volume in all wells is 100 µL.

Transfer

100

µL

Reconstituted, diluted

Rat IL-4 Standard

Sample Diluent

100 µL

Discard

100 µL

Fig. 1 Dilute standards - microwell plate

3. Add 100 µL of Sample Diluent in duplicate to blank wells.

4. Add 50 µL of Sample Diluent to the sample wells.

5. Add 50 µL of each sample in duplicate to the sample wells.

6. Prepare Biotin-Conjugate (see “Preparation of Biotin-

Conjugate“ on page 2)

7. Add 50 µL of prepared Biotin-Conjugate to all wells.

8. Cover with an adhesive lm and incubate at room temperature

(18°C to 25°C) for 2 hours, on a microplate shaker if available.

9. Prepare Streptavidin-HRP (see “Preparation of Streptavidin-

HRP“ on page 2).

10. Remove adhesive lm and empty wells. Wash microwells 3 times

according to point a. of the test protocol. Proceed immediately to

the next step.

11. Add 100 µL of diluted Streptavidin-HRP to all wells, including

the blank wells.

12. Cover with an adhesive lm and incubate at room temperature

(18°C to 25°C) for 1 hour, on a microplate shaker if available.

13. Remove adhesive lm and empty wells. Wash microwells 3 times

according to point a. of the test protocol. Proceed immediately to

the next step.

14. Pipee 100 µL of Substrate Solution to all wells.

15. Incubate the microwell strips at room temperature (18°C to 25°C)

for about 10 minutes. Avoid direct exposure to intense light.

Monitor the color development on the plate. The substrate

reaction should be stopped (see next point of this protocol) before

positive wells are no longer properly recordable.

Determination of the ideal time period for color development has

to be done individually for each assay.

Add the stop solution when the highest standard has developed a

dark blue color. Alternatively the color development can be

monitored on a plate reader at 620 nm. The substrate reaction

should be stopped as soon as Standard 1 has reached an OD of 0.9

– 0.95.

Rat IL-4 Matched Pair User Guide

16. Stop the enzyme reaction by quickly pipeing 100 µL of Stop

Solution into each well. It is important that the Stop Solution is

spread quickly and uniformly throughout the microwells to

completely inactivate the enzyme. Results must be read

immediately after the Stop Solution is added, or within one hour

if the microwell strips are stored at 2°C to 8°C in the dark.

17. Read absorbance of each microwell on a spectro-photometer

using 450 nm as primary wave length (you can use 620 nm as

reference wave length; 610 nm to 650 nm is acceptable). Blank the

plate reader according to the manufacturer's instructions by using

the blank wells. Determine the absorbance of both the samples

and the rat IL-4 standards.

Calculation of results

• Calculate the average absorbance values for each set of duplicate

standards and samples. Duplicates should be within 20% of the

mean value.

• Create a standard curve by ploing the mean absorbance for each

standard concentration on the y-axis, against the rat IL-4

concentration on the x-axis. Draw a best t curve through the

points of the graph (a 5-parameter curve t is recommended).

• To determine the concentration of soluble rat IL-4 for each

sample, rst calculate the mean absorbance value for the

duplicate wells of the sample, then extend a horizontal line from

this point on the y-axis to the standard curve. At the point of

intersection, extend a vertical line to the x-axis and read the

corresponding rat IL-4 concentration.

• If instructions in this protocol have been followed samples have

been diluted 1:2 (50 µL sample + 50 µL Sample Diluent), the

concentration read from the standard curve must be multiplied

by the dilution factor (x2).

• Calculation of samples with a concentration exceeding that of

standard 1 may result in inaccurate, low rat IL-4 levels. Such

samples require further external predilution according to

expected rat IL-4 values with Sample Diluent in order to precisely

quantitate the actual rat IL-4 level.

• Each testing facility should establish a control sample of known

rat IL-4 concentration and run this additional control with each

assay. If the values obtained are not within the expected range of

this control, the assay results may be invalid.

A basic understanding of immunoassay development and technical

experience in ELISA performance are conditional for the successful

use of this Module Set.

The protocol provided is just a guideline. The type of substrate as well

as all other reagents not included in the Module Set may inuence the

test characteristics.

Rat IL-4 module set characteristics

Speciﬁcity

The interference of circulating factors of the immune system was

evaluated by spiking these proteins at physiologically relevant

concentrations into a rat IL-4 positive serum. There was no cross

reactivity detected.

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Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria

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The information in this guide is subject to change without notice.

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23 August 2019

Thermo Fisher Scientific Rat IL-4 Matched Pair User guide

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